DOCSLIB.ORG

Immunodeficiency Virus Genome Affects Virus Production and Infectivity T

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Immunodeficiency Virus Genome Affects Virus Production and Infectivity T Proc. Natl. Acad. Sci. USA Vol. 89, pp. 4114-4118, May 1992 Microbiology Mutation in the primer binding site of the type 1 human immunodeficiency virus genome affects virus production and infectivity T. NAGASHUNMUGAM*, A. VELPANDI*, C. S. GOLDSMITHt, S. R. ZAKIt, V. S. KALYANARAMANt, AND A. SRINIVASAN*§ *The Wistar Institute of Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19104; tDivision of Viral and Rickettsial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA 30333; and tAdvanced Bioscience Laboratories, Inc., 5510 Nicholson Lane, Kensington, MD 20805 Communicated by Hilary Koprowski, January 16, 1992 ABSTRACT In an effort to understand the contribution of retroviruses make use of tryptophan tRNA. Sequence anal- the primer-binding site (PBS) region to human immunodefi- ysis of HIV-1 revealed PBS corresponding to the lysine ciency virus (HIV) replication, we have constructed a mutant tRNA as in other lentiviruses (7). It has also been recently HIV proviral DNA with an alteration in the 5' end of the PBS. demonstrated that HIV RT forms a stable complex with The PBS mutant proviral DNA was characterized by transfec- isoacceptor 3 of lysine tRNA (Lys3 tRNA) (8), as has been tion of the viral DNA into CD4' and non-CD4' target cells. demonstrated for the RT enzyme of avian viruses. The results indicate that mutation in the PBS reduced the level Initiation of viral DNA synthesis is a crucial step in the of viral particles released into the medium of transfected cells replication of retroviruses, and the essential PBS is embed- in comparison to wild-type proviral DNA. The viral particles ded in the complex structure ofRNA (9). A number ofstudies were noninfectious upon transmission to established CD4' cell have reported the importance of the secondary structure of lines and phytohemagglutinin-stimulated peripheral blood the genome at the 5' end in the assembly and infectivity ofthe lymphocytes. Electron microscopic analysis of the transfected virus (10-12). In an effort to understand the structural cells revealed no abnormalities in the structure of the virion requirement of the PBS in reverse transcription and virus directed by the mutant proviral DNA. Also, the protein and morphogenesis, we have introduced an insertional mutation RNA contents of the mutant virions were similar to the wild in the PBS of HIV-1. The results obtained with the PBS type. The quantitation of intracellular viral structural protein mutant imply that the PBS region, in addition to its partici- in the transfected cells, however, indicated that the PBS pation in reverse transcription, is also involved in the assem- mutation may have an effect on the assembly of viral particles bly of viral particles. in addition to completely abolishing reverse transcription of viral RNA into DNA. These results provide evidence that the MATERIALS AND METHODS PBS region of the viral genome has multiple functions in HIV-I Cell Lines. A human rhabdomyosarcoma (RD) cell line replication. (American Type Culture Collection) was maintained as a monolayer culture in Dulbecco's modified Eagle's medium The genome of human immunodeficiency virus type 1 (HIV- supplemented with 10% fetal bovine serum, penicillin (100 1), like other members of the lentivirus family, encodes not units/ml), and L-glutamine (540 pug/ml) at 370C with 5% CO2. only the virion proteins gag, pol, and env common to all CEM x 174 and HUT78 cells were maintained as suspension replication-competent retroviruses but also eight accessory cultures in RPMI 1640 medium; phytohemagglutinin- genes, some of which are involved in the gene regulation and stimulated (10 Ag/ml) peripheral blood lymphocytes were morphogenesis of HIV (1-3). Biochemical and structural grown in RPMI 1640 medium containing T-cell growth factor studies have revealed that HIV is about 80-120 nm in (10%). diameter and consists of an inner core (nucleoid) surrounded Construction of the PBS Mutant. The HIV proviral DNA by an outer envelope. The core corresponds to a ribonucleo- plasmid designated pARV was derived from cells infected protein surrounded by the capsid protein. The viral RNA with HIVSF2 (13). The pARV plasmid was cleaved with the genome is in the ribonucleoprotein, and it is composed oftwo restriction enzyme Nar I, and the resultant linear molecule identical positive-sense RNAs with a 5' cap and a 3' poly(A) was purified by agarose gel electrophoresis. The Nar I structure (4). overhang sequence in the DNA was filled in by using Klenow The replication cycle of HIV-1 and other retroviruses polymerase and ligated by using T4 ligase (14). The recom- involves the reverse transcription of virion RNA to viral binant plasmid obtained from the transformed colonies was DNA, which then becomes incorporated into the host cellular checked for the presence of the Nar I cleavage site. The genome. The reverse transcription is mediated by the virion- plasmid (pARV-NAR) lacking the Nar I site was further associated enzyme reverse transcriptase (RT) (5, 6). As with confirmed by sequence analysis with a Sequenase kit (United all DNA polymerases, RT needs a primer covering a 3' States Biochemical). hydroxyl (OH) group to initiate cDNA synthesis. The in vivo Transfection. RD cells (1 x 106 cells per plate) were split 24 primer for the viral first-strand cDNA synthesis from an RNA hr before transfection, and growth medium was replaced 1-2 template has been shown to be tRNA (6). A region near the hr before the addition of calcium phosphate-precipitated 5' end of the viral RNA genome designated as the primer- DNA (15). Cells were exposed to the precipitate for 8 hr binding site (PBS) is complementary to the 18 nucleotides of followed by a 90-sec glycerol shock (16). For DEAE-dextran the 3' CCA end ofthe specific tRNA primer. Predominantly, transfection, CEM x 174 cells (1 x 107) were washed and many mammalian retroviruses use proline tRNA, and avian Abbreviations: RT, reverse transcriptase; PBS, primer-binding site; The publication costs of this article were defrayed in part by page charge RD, rhabdomyosarcoma; HIV-1, human immunodeficiency virus payment. This article must therefore be hereby marked "advertisement" type 1; RIPA, radioimmunoprecipitation analysis. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 4114 Downloaded by guest on September 24, 2021 Microbiology: Nagashunmugam et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4115 PBS the PBS was confirmed by restriction enzyme and sequence LTR GAG POt ENV LEF analyses. Biological Characterization of HIV-1 Proviral DNA with an Altered PBS. To test the mutant for replication competence, TGGCGCCCGAACAGGGAC pARV and mutant proviral DNAs were directly trans- Nar I wild-type fected into a CD4' cell line designated CEM X 174. A TGGCGcgCCCGAACAGGGAC pARV-NJAR positive result is indicative of a spreading HIV-1 infection in this CD4' cell line. Transfected CEM x 174 cells with FIG. 1. Structure of wild-type and Imutated proviral DNAs used wild-type proviral DNA showed high levels of HIV-1 p24 in this study. The wild-type PBS seque nce is shown. The insertion antigen in the medium, in comparison to the PBS mutant mutation in the PBS region of the muitant proviral DNA (pARV- (Table 1). These results indicate that virus particles directed NAR) is indicated in lowercase letters. LTR, long terminal repeat. by the mutant proviral DNA are unable to initiate a spreading infection. .iumcontainin100 of suspended in 1 ml of serum-free medium containing 100,.ag of Effect of the PBS Mutation on Virus Production. To distin- DEAE-dextran and 10 ,ug ofplasmi( iDNA for 1-2 hr inaPetri guish the effect of the mutation production (synthesis dish. The cells were washed once and were grown in RPMI with 109woserum (17). and assembly of viral particles), we have carried out trans- fection experiments using a transient expression system assay (Coul- HIV Antigen and RT Assay. The HIV antigen(17).en assay (Coul- involving monolayer cells. Human RD cells ideal for ter) was performed according to t these studies, because they have been shown to release high lines to quantitate the amount of herusmanufacTer'assguid procedure was essentially similar to oruat(d8)criTbedR(T asThe levels of viral particles upon transfection as monitored by RT oi and a viral antigen assay (24-26). Transfection of the mutant endogenous RT reaction was carrierd outthatdescribsedn(9In the absence ofofthethe proviral DNA consistently showed a 40-60% reduction in the exogenous template poly(A)-(dT)12 Immunoprecipitation Analysis. i 1D cells102)(2 x trans- release of viral particles in comparison to wild type. The fected with 10 of or 10 IDgof mutant pARV-NAR differences observed in the extracellular viral p24 prompted jig pARV , us agtof mutantol Th to quantitate the intracellular p24 in cells transfected with proviral DNA were labeled 60 hr after glycerol shock. The mutant and wild-type proviral DNA (Table 2). Unlike extra- labeling continued for 12 hr with 8 ml of methionine- and cellular p24 levels, intracellular p24 levels similar in cysteine-deficient serum-free mediu m containinge50r Ciu( Ci both wild-type and mutant proviral DNA-transfected cells = 37 GBq) of Tran35S-label per ml. The cell-free medium and (Table 3). (al ) cells were analyzed by using HIV--positive patient sera and The viral particles released from the transfected cultures normal human sera (21). werre fixed for EM with 2.5% infectivity potential. Since our previous EM. After transfection, cells emixm edtfordEM studies (27) and others (28) have shown that the proviral clone glutaraldehyde, postfixed with osi tetroxide, and em- pARV-derived virus is not able to infect CEM 174 cells bedded in Sectionisum Epon/Araldite. a cell-free virus, we have used the cocultivation method to citrate and uranyl acetate (22).
Load more

Recommended publications
  • Formation of Virus-Like Particles from Human Cell Lines Exclusively Expressing Influenza Neuraminidase
    Journal of General Virology (2010), 91, 2322–2330 DOI 10.1099/vir.0.019935-0 Formation of virus-like particles from human cell lines exclusively expressing influenza neuraminidase Jimmy C. C. Lai,1 Wallace W. L. Chan,1 Franc¸ois Kien,1 John M. Nicholls,2 J. S. Malik Peiris1,3 and Jean-Michel Garcia1 Correspondence 1HKU-Pasteur Research Centre, Hong Kong SAR Jean-Michel Garcia 2Department of Pathology, The University of Hong Kong, Hong Kong SAR [email protected] 3Department of Microbiology, The University of Hong Kong, Hong Kong SAR The minimal virus requirements for the generation of influenza virus-like particle (VLP) assembly and budding were reassessed. Using neuraminidase (NA) from the H5N1 and H1N1 subtypes, it was found that the expression of NA alone was sufficient to generate and release VLPs. Biochemical and functional characterization of the NA-containing VLPs demonstrated that they were morphologically similar to influenza virions. The NA oligomerization was comparable to that of the live virus, and the enzymic activity, whilst not required for the release of NA-VLPs, was Received 8 January 2010 preserved. Together, these findings indicate that NA plays a key role in virus budding and Accepted 23 May 2010 morphogenesis, and demonstrate that NA-VLPs represent a useful tool in influenza research. INTRODUCTION membrane. HA binds to sialic acid receptors on the cell surface and mediates the fusion process (Matrosovich et al., Influenza A viruses are lipid-enveloped members of the 2006), whereas NA cleaves the terminal sialic acids from family Orthomyxoviridae. They contain eight negative-sense, the cell-surface glycans to facilitate release of the progeny single-stranded RNA segments encoding ten viral proteins.
    [Show full text]
  • Intracellular Trafficking of HBV Particles
    cells Review Intracellular Trafficking of HBV Particles Bingfu Jiang 1 and Eberhard Hildt 1,2,* 1 Department of Virology, Paul-Ehrlich-Institut, D-63225 Langen, Germany; [email protected] 2 German Center for Infection Research (DZIF), TTU Hepatitis, Marburg-Gießen-Langen, D63225 Langen, Germany * Correspondence: [email protected]; Tel.: +49-61-0377-2140 Received: 12 August 2020; Accepted: 2 September 2020; Published: 2 September 2020 Abstract: The human hepatitis B virus (HBV), that is causative for more than 240 million cases of chronic liver inflammation (hepatitis), is an enveloped virus with a partially double-stranded DNA genome. After virion uptake by receptor-mediated endocytosis, the viral nucleocapsid is transported towards the nuclear pore complex. In the nuclear basket, the nucleocapsid disassembles. The viral genome that is covalently linked to the viral polymerase, which harbors a bipartite NLS, is imported into the nucleus. Here, the partially double-stranded DNA genome is converted in a minichromosome-like structure, the covalently closed circular DNA (cccDNA). The DNA virus HBV replicates via a pregenomic RNA (pgRNA)-intermediate that is reverse transcribed into DNA. HBV-infected cells release apart from the infectious viral parrticle two forms of non-infectious subviral particles (spheres and filaments), which are assembled by the surface proteins but lack any capsid and nucleic acid. In addition, naked capsids are released by HBV replicating cells. Infectious viral particles and filaments are released via multivesicular bodies; spheres are secreted by the classic constitutive secretory pathway. The release of naked capsids is still not fully understood, autophagosomal processes are discussed. This review describes intracellular trafficking pathways involved in virus entry, morphogenesis and release of (sub)viral particles.
    [Show full text]
  • A SARS-Cov-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing
    A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing Supplementary Information Supplementary Discussion All SARS-CoV-2 protein and gene functions described in the subnetwork appendices, including the text below and the text found in the individual bait subnetworks, are based on the functions of homologous genes from other coronavirus species. These are mainly from SARS-CoV and MERS-CoV, but when available and applicable other related viruses were used to provide insight into function. The SARS-CoV-2 proteins and genes listed here were designed and researched based on the gene alignments provided by Chan et. al. 1 2020 .​ Though we are reasonably sure the genes here are well annotated, we want to note that not every ​ protein has been verified to be expressed or functional during SARS-CoV-2 infections, either in vitro or in vivo. ​ ​ In an effort to be as comprehensive and transparent as possible, we are reporting the sub-networks of these functionally unverified proteins along with the other SARS-CoV-2 proteins. In such cases, we have made notes within the text below, and on the corresponding subnetwork figures, and would advise that more caution be taken when examining these proteins and their molecular interactions. Due to practical limits in our sample preparation and data collection process, we were unable to generate data for proteins corresponding to Nsp3, Orf7b, and Nsp16. Therefore these three genes have been left out of the following literature review of the SARS-CoV-2 proteins and the protein-protein interactions (PPIs) identified in this study.
    [Show full text]
  • Symposium on Viral Membrane Proteins
    Viral Membrane Proteins ‐ Shanghai 2011 交叉学科论坛 Symposium for Advanced Studies 第二十七期：病毒离子通道蛋白的结构与功能研讨会 Symposium on Viral Membrane Proteins 主办单位：中国科学院上海交叉学科研究中心 承办单位：上海巴斯德研究所 1 Viral Membrane Proteins ‐ Shanghai 2011 Symposium on Viral Membrane Proteins Shanghai Institute for Advanced Studies, CAS Institut Pasteur of Shanghai,CAS 30.11. – 2.12 2011 Shanghai, China 2 Viral Membrane Proteins ‐ Shanghai 2011 Schedule: Wednesday, 30th of November 2011 Morning Arrival Thursday, 1st of December 2011 8:00 Arrival 9:00 Welcome Bing Sun, Co-Director, Pasteur Institute Shanghai 9: 10 – 9:35 Bing Sun, Pasteur Institute Shanghai Ion channel study and drug target fuction research of coronavirus 3a like protein. 9:35 – 10:00 Tim Cross, Tallahassee, USA The proton conducting mechanism and structure of M2 proton channel in lipid bilayers. 10:00 – 10:25 Shy Arkin, Jerusalem, IL A backbone structure of SARS Coronavirus E protein based on Isotope edited FTIR, X-ray reflectivity and biochemical analysis. 10:20 – 10:45 Coffee Break 10:45 – 11:10 Rainer Fink, Heidelberg, DE Elektromechanical coupling in muscle: a viral target? 11:10 – 11:35 Yechiel Shai, Rehovot, IL The interplay between HIV1 fusion peptide, the transmembrane domain and the T-cell receptor in immunosuppression. 11:35 – 12:00 Christoph Cremer, Mainz and Heidelberg University, DE Super-resolution Fluorescence imaging of cellular and viral nanostructures. 12:00 – 13:30 Lunch Break 3 Viral Membrane Proteins ‐ Shanghai 2011 13:30 – 13:55 Jung-Hsin Lin, National Taiwan University Robust Scoring Functions for Protein-Ligand Interactions with Quantum Chemical Charge Models. 13:55 – 14:20 Martin Ulmschneider, Irvine, USA Towards in-silico assembly of viral channels: the trials and tribulations of Influenza M2 tetramerization.
    [Show full text]
  • Opportunistic Intruders: How Viruses Orchestrate ER Functions to Infect Cells
    REVIEWS Opportunistic intruders: how viruses orchestrate ER functions to infect cells Madhu Sudhan Ravindran*, Parikshit Bagchi*, Corey Nathaniel Cunningham and Billy Tsai Abstract | Viruses subvert the functions of their host cells to replicate and form new viral progeny. The endoplasmic reticulum (ER) has been identified as a central organelle that governs the intracellular interplay between viruses and hosts. In this Review, we analyse how viruses from vastly different families converge on this unique intracellular organelle during infection, co‑opting some of the endogenous functions of the ER to promote distinct steps of the viral life cycle from entry and replication to assembly and egress. The ER can act as the common denominator during infection for diverse virus families, thereby providing a shared principle that underlies the apparent complexity of relationships between viruses and host cells. As a plethora of information illuminating the molecular and cellular basis of virus–ER interactions has become available, these insights may lead to the development of crucial therapeutic agents. Morphogenesis Viruses have evolved sophisticated strategies to establish The ER is a membranous system consisting of the The process by which a virus infection. Some viruses bind to cellular receptors and outer nuclear envelope that is contiguous with an intri‑ particle changes its shape and initiate entry, whereas others hijack cellular factors that cate network of tubules and sheets1, which are shaped by structure. disassemble the virus particle to facilitate entry. After resident factors in the ER2–4. The morphology of the ER SEC61 translocation delivering the viral genetic material into the host cell and is highly dynamic and experiences constant structural channel the translation of the viral genes, the resulting proteins rearrangements, enabling the ER to carry out a myriad An endoplasmic reticulum either become part of a new virus particle (or particles) of functions5.
    [Show full text]
  • Coronavirus Proteins As Ion Channels: Current and Potential Research
    Washington University School of Medicine Digital Commons@Becker Open Access Publications 1-1-2020 Coronavirus proteins as ion channels: Current and potential research Conor McClenaghan Alex Hanson Sun-Joo Lee Colin G Nichols Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs REVIEW published: 09 October 2020 doi: 10.3389/fimmu.2020.573339 Coronavirus Proteins as Ion Channels: Current and Potential Research Conor McClenaghan, Alex Hanson, Sun-Joo Lee and Colin G. Nichols* Center for Investigation of Membrane Excitability Diseases, and Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, United States Coronavirus (CoV) outbreaks have recently emerged as a global public health threat due to their exceptional zoonotic potential — a feature arising from their ability to infect a diverse range of potential hosts combined with their high capacity for mutation and recombination. After Severe Acute Respiratory Syndrome (SARS) CoV-1 in 2003 and Middle East Respiratory Syndrome (MERS) CoV in 2012, with the current SARS-CoV-2 pandemic we are now in the midst of the third deadly international CoV outbreak in less than 20 years. Coronavirus outbreaks present a critical threat to global public health and Edited by: Julia Kzhyshkowska, an urgent necessity for therapeutic options. Here, we critically examine the current Heidelberg University, Germany evidence for ion channel activity in CoV proteins and the potential for modulation as a Reviewed by: therapeutic approach. Jaume Torres, Nanyang Technological University, Keywords: Severe Acute Respiratory Syndrome coronavirus-2, ion channel, spike protein, electrophysiology, Singapore bilayer, Severe Acute Respiratory Syndrome coronavirus Srinivasa Reddy Bonam, Institut National de la Sante´ et de la Recherche Me´ dicale (INSERM), France INTRODUCTION *Correspondence: Coronaviruses (CoVs) are enveloped, single-stranded, positive-sense RNA viruses that were first Colin G.
    [Show full text]
  • Function and Structure of Viral Ribonucleoproteins Complexes”
    viruses Editorial Special Issue “Function and Structure of Viral Ribonucleoproteins Complexes” Serena Bernacchi Architecture et Réactivité de l’ARN-CNRS UPR 9002, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire, 67084 Strasbourg, France; [email protected] Academic Editor: Eric Freed Received: 23 November 2020; Accepted: 24 November 2020; Published: 26 November 2020 RNA viruses are extraordinary evolution machines that efficiently ensure their replication by taking advantage of the association with viral and cellular components to form ribonucleic complexes (vRNPs). These vRNPs are functional units of the infectious cycle, driving various processes including transcription, nuclear export, translation, and intracellular trafficking pathways for targeting viral components to the assembly sites where the packaging of the viral RNA genetic material into virions occurs. The aim of this Special Issue of Viruses is to provide an updated picture of the structural organization and of the different roles of vRNPs in various viral systems. The first part of the SI is dedicated to HIV-1 which is the causative agent of AIDS. Since its discovery in 1983, HIV-1 has become one of the leading causes of death worldwide. Up to now, the pandemic persists despite the implementation of highly active antiretroviral therapy, and over the years, a vast spectrum of techniques has been implemented to diagnose and monitor AIDS progression. Besides the conventional approaches, recently, microfluidics has provided useful methods for monitoring HIV-1 infection. This powerful tool allows information at the single-cell scale and high-throughput development. Herein, Eid et al. highlighted recent significant advances of continuous microfluidics in AIDS diagnosis, and in the basic study of the HIV-1 life cycle [1].
    [Show full text]
  • Protein Transduction by Pseudotyped Lentivirus-Like Nanoparticles
    Gene Therapy (2011) 18, 936–941 & 2011 Macmillan Publishers Limited All rights reserved 0969-7128/11 www.nature.com/gt SHORT COMMUNICATION Protein transduction by pseudotyped lentivirus-like nanoparticles T Aoki1,2, K Miyauchi1,2, E Urano1, R Ichikawa1 and J Komano1 A simple, efficient and reproducible method to transduce proteins into mammalian cells has not been established. Here we describe a novel protein transduction method based on a lentiviral vector. We have developed a method to package several thousand foreign protein molecules into a lentivirus-like nanoparticle (LENA) and deliver them into mammalian cells. In this proof-of-concept study, we used b-lactamase (BlaM) as a reporter molecule. The amino-terminus of BlaM was fused to the myristoylation signal of lyn, which was placed upstream of the amino-terminus of Gag (BlaM-gag-pol). By co-transfection of plasmids encoding BlaM-gag-pol and vesicular stomatitis virus-G (VSV-G) into 293T cells, LENA were produced containing BlaM enzyme molecules as many as Gag per capsid, which has been reported to be B5000 molecules, but lacking the viral genome. Infection of 293T and MT-4 cells by VSV-G-pseudotyped BlaM-containing LENA led to successful transduction of BlaM molecules into the cell cytoplasm, as detected by cleavage of the fluorescent BlaM substrate CCF2-AM. LENA-mediated transient protein transduction does not damage cellular DNA, and the preparation of highly purified protein is not necessary. This technology is potentially useful in various basic and clinical applications. Gene Therapy (2011) 18, 936–941; doi:10.1038/gt.2011.38; published online 31 March 2011 Keywords: LENA; BlaM; Gag; protein transduction; lentiviral vector INTRODUCTION cells.
    [Show full text]
  • Persistence of HIV-1 Structural Proteins and Glycoproteins in Lymph Nodes of Patients Under Highly Active Antiretroviral Therapy
    Persistence of HIV-1 structural proteins and glycoproteins in lymph nodes of patients under highly active antiretroviral therapy Mikulas Popovic*†, Klara Tenner-Racz‡, Colleen Pelser*, Hans-Jurgen Stellbrink§, Jan van Lunzen§, George Lewis*, Vaniambadi S. Kalyanaraman¶, Robert C. Gallo*†, and Paul Racz‡ *Institute of Human Virology, University of Maryland Biotechnology Institute, University of Maryland, Baltimore, MD 21201; ‡Bernhard-Nocht Institute for Tropical Diseases, 2000 Hamburg 4, Germany; §University Hospital Eppendorf, D-20246 Hamburg, Germany; and ¶Advanced BioScience Laboratories, Kensington, MD 20895 Contributed by Robert C. Gallo, August 12, 2005 Here we report a long-term persistence of HIV-1 structural proteins a slow and incomplete process in a number of long-term treated and glycoproteins in germinal centers (GCs) of lymph nodes (LNs) patients (19–22). in the absence of detectable virus replication in patients under Earlier in vitro studies explored interactions of mononuclear cells highly active antiretroviral therapy (HAART). The persistence of from peripheral blood with native or recombinant HIV-1 structural viral structural proteins and glycoproteins in GCs was accompanied proteins and glycoproteins, with the matrix protein HIV-1p17 (23, by specific antibody responses to HIV-1. Seven patients during the 24), and particularly with the HIV-1Env (gp120͞160) (25). These chronic phase of HIV-1 infection were analyzed for the presence of extensive studies of B and T cell interactions with HIV-1Env and the capsid protein (HIV-1p24), matrix protein (HIV-1p17), and HIV-1p17 showed a broad spectrum of changes in cell surface envelope glycoproteins (HIV-1gp120͞gp41), as well as for viral RNA markers, cytokine production, B cell maturation, and increased T (vRNA) in biopsy specimens from LNs obtained before initiation of cell proliferation and HIV-1 replication in the virus-infected T cell therapy and during HAART that lasted from 5 to 13 months.
    [Show full text]
  • Characterization of Molecular Targets for Differential Regulation of the Type I and III Interferon Induction and Signalling Pathways by Rotavirus NSP1
    Characterization of molecular targets for differential regulation of the type I and III interferon induction and signalling pathways by rotavirus NSP1 Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Gennaro Iaconis September 2018 Table of contents TABLE OF CONTENTS ............................................................................................................................... 1 LIST OF FIGURES ...................................................................................................................................... 6 LIST OF TABLES ........................................................................................................................................ 8 DECLARATION ....................................................................................................................................... 10 ABSTRACT ............................................................................................................................................. 11 1 INTRODUCTION ........................................................................................................................... 12 1.1 Rotavirus ................................................................................................................. 12 1.1.1 Historical Background ......................................................................................... 12 1.1.2 Classification ......................................................................................................
    [Show full text]
  • Rev-Induced Modulation of Nef Protein Underlies Temporal Regulation of Human Immunodeficiency Virus Replication NAFEES AHMAD, RATAN K
    Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6111-6115, August 1989 Biochemistry Rev-induced modulation of Nef protein underlies temporal regulation of human immunodeficiency virus replication NAFEES AHMAD, RATAN K. MAITRA, AND SUNDARARAJAN VENKATESAN* Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Bernard Moss, May 25, 1989 ABSTRACT The replication of human immunodeficiency of Nef expressed from a Rev- provirus that was nonrepli- virus type 1 (HIV-1) requires the concerted action of two cating and had repressed Tat function. Rev-induced loss of virus-encoded transactivator proteins, Tat and Rev, and is in Nef synthesis paralleled augmented expression of viral pro- turn moderated by the viral transcriptional repressor Nef. We teins, and the magnitude of the Rev effect could be reduced show here that the phenotype of a Rev- HIV-1 provirus was by addition of exogenous Nef. nonreplicating and was distinguished by accumulation of Nef protein and reduced Tat function. Provirus defective in both the rev and nefgenes (Rev-Nef ) was also nonreplicating but MATERIALS AND METHODS had normal Tat function. Trans-complementation of the Rev- Virus and Cell Cultures. CD4' (Leu-3+) A3.01 T lympho- mutant with Rev caused a decrease ofboth the steady-state level cytes (21) in RPMI 1640 medium with 10% fetal bovine serum and the rate of synthesis of Nef. This was accompanied by were used for infection. For most DNA transfections, SW480 enhanced synthesis of viral structural proteins. Rev induced (22) or HeLa S3 monolayers or A3.01 cells were used.
    [Show full text]
  • Induction of Cell Death in the Human Acute Lymphoblastic Leukemia Cell Line Reh by Infection with Rotavirus Isolate Wt1-5
    biomedicines Article Induction of Cell Death in the Human Acute Lymphoblastic Leukemia Cell Line Reh by Infection with Rotavirus Isolate Wt1-5 Rafael Guerrero , Carlos Guerrero * and Orlando Acosta Department of Physiological Sciences, Faculty of Medicine, Universidad Nacional de Colombia, Carrera 30 No. 45-03 Bloque 47, Ciudad Universitaria, Bogotá 111321, Colombia; [email protected] (R.G.); [email protected] (O.A.) * Correspondence: [email protected]; Tel.: +57-1-3165000 (ext. 15052-15053) Received: 17 June 2020; Accepted: 23 June 2020; Published: 24 July 2020 Abstract: Cancer is a major health problem that poses a great challenge to health care systems worldwide. Tools for cancer treatment have rapidly advanced in recent years, resulting in therapeutic strategies which are alternative and complementary to conventional treatment. To identify the cell surface receptors used by a tumor cell-adapted rotavirus and the cell death markers induced by its infection, we use Wt1-5, a rotavirus isolate recently adapted to tumor cells, to infect the human acute lymphoblastic leukemia cell line, Reh. The expression of cell surface receptors used by Wt1-5 was determined using flow cytometry and an antibody blocking assay to test for their implication in virus infection. Viral antigens and cell death markers induced by rotavirus infection were followed by flow cytometric analysis. The present study showed that rotavirus Wt1-5 was able to use cell surface proteins such as heat shock proteins (HSPs) 90, 70, 60 and 40, Hsc70, PDI and integrin β3. Rotavirus Wt1-5 induced cytotoxic effects including changes in cell membrane permeability, alteration of mitochondrial membrane potential, DNA fragmentation and activation of cell death signaling.
    [Show full text]